Isolation of cells for in vitro studies or for applications in cellular therapies usually requires an initial separation of blood cell components mainly based on the bulk depletion of erythrocytes, which comprise >99% of the cellular mass of blood. These techniques for erythrocyte removal are based on hypotonic lysis of erythrocytes, density gradient separation, or enhanced centrifugal sedimentation using heta starch. Hypotonic lysis, while useful in low volume in vitro studies, is inefficient and impractical for the large volumes of blood tissues processed for cellular therapies.
Density-gradient separation relies on small differences in the density of different cell types causing them to segregate at different levels in a fluid medium of variable density. Differences in density between the cell types can be small, and individual cells types can be heterogeneous in size and density. Consequently, particular cell types can become distributed throughout a density-gradient medium rather than precisely segregating at a discrete area in the density medium, resulting in poor recovery of desired cells and/or contamination with undesired cell types. In procedures that enrich for rare blood cell types such as hematopoietic progenitor cells, density-gradient sedimentation generally results in poor yields. For example, using conventional density-gradient methods to isolate progenitor cells (e.g., CD34+ hematopoietic stem cells) from umbilical cord blood reportedly results in a significant loss of the desired stem cells. See e.g., Wagner, J. E., Am J Ped Hematol Oncol 15:169 (1993). As another example, using conventional density-gradient methods to isolate lymphocytes reportedly results in selective loss of particular lymphocyte subsets. See e.g., Collins, D. P., J Immunol Methods 243:125 (2000).
An additional method for removing erythrocytes from blood includes using heta starch, which stimulates the formation of erythrocyte aggregates that sediment more rapidly than leukocyte components when sedimented at 50×g in a centrifuge. While this method is non-toxic and ‘safe’ for the recipient, its performance in the recovery of important cell types (e.g., hematopoietic stem cells) is highly variable depending upon factors such as temperature, age of sample (post-collection) prior to processing, cellularity of sample, and volume of sample. These factors, with respect to umbilical cord blood, for example, can often result in poor recovery of stem cells and diminution of the engraftment potential of the cord blood cells, increasing the risk for transplant failure.
Increasing the recovery of rare cell types from donor tissue could dramatically improve the success of transplant and immune therapies (e.g., bone marrow transplants, stem cell-based gene therapy, and immune cell therapy), the success of which apparently is related to the actual number of the cells being used for therapy.